The specific recognition of an HLA/peptide complex by a T cell through the T cell receptor (TCR) is a crucial step in initiating an immune response, whether this response is normal, as in the recognition of foreign antigen, or abnormal, as occurs in the initiation of autoimmunity. The specificity conferred on an immune response by the TCR has made it an increasingly popular target for novel therapeutic intervention strategies intended to specifically interrupt autoimmune responses. Despite the appealing nature of TCRs as possible targets for therapy, comparatively little data is available regarding the specific mechanics of the interaction between TCRs and the complex ligand formed by an HLA molecule and a peptide antigen. Most mutagenesis studies of the structure of the trimolecular complex have focused on peptides, which ar relatively easy to alter at any position, or on HLA molecules which exist in numerous different allelic forms and have been further altered by in vitro mutagenesis in some studies. In order to dissect the molecular recognition of HLA/peptide complexes by TCR, and, in particular, to define residues of the TCR that interact with HLA-DR, we have chosen to study the DR restricted recognition of the immunodominant peptide of influenza virus hemagglutinin, HA 307-319, by site directed mutagenesis of TCR genes and their re-introduction into appropriate recipient T cell lines. In preliminary experiments, w have isolated a number of HA 307-319 specific T cell clones restricted by various DR molecules, developed an efficient system for mutagenesis and re-expression.of TCR genes, obtained antigen specific activation of transfected TCRs, and have begun to carry out chain swapping and domain shuffling experiments that should map the location of some potential contact sites for HLA-DR as well as peptide on the TCR molecule. In this application, we propose to build upon these promising preliminary results by (1) expanding our panel of HA 307-319 specific T cell clones to include clones of additional DR restrictions, (2) carry out a systematic probing of the structurally important features of the TCRs from these clones by site directed mutagenesis, and (3) develop an in vitro system for randomly mutagenizing TCR genes, and selecting for novel receptors with altered HLA-DR restriction or antigen specificity. There is a distinct need for structural studies of this nature in human model systems in order to be able to make rational decisions about how to design specific peptide inhibitors, such as TCR antagonists, that could be used therapeutically in autoimmune diseases.